Tumor necrosis factor-.alpha. (TNF-.alpha., also known as cachectin) is a mammalian protein capable of inducing a variety of effects on numerous cell types. TNF-.alpha. was initially characterized by its ability to cause lysis of tumor cells and is produced by activated cells such as mononuclear phagocytes, T-cells, B-cells, mast cells and NK cells. In mononuclear phagocytes, TNF-.alpha. is initially synthesized as a membrane-bound protein of approximately 26 kD. A 17 kD fragment of the 26 kD membrane-bound TNF-.alpha. is "secreted" and combines with two other secreted TNF-.alpha. molecules to form a circulating 51 kD homotrimer. TNF-.alpha. is a principal mediator of the host response to gram-negative bacteria. Lipopolysaccharide (LPS, also called endotoxin), derived from the cell wall of gram-negative bacteria, is a potent stimulator of TNF-.alpha. synthesis. Because the deleterious effects which can result from an over-production or an unregulated-production of TNF are extremely serious, considerable efforts have been made to control or regulate the serum level of TNF. An important part in the effort to effectively control serum TNF levels is the understanding of the mechanism of TNF biosynthesis.
The mechanism by which TNF-.alpha. is secreted has only been recently elucidated. Kriegler et al. Cell, 53, 45-53, (1988) conjectured that TNF-.alpha. "secretion" is due to the cleaving of the 26 kD membrane-bound molecule by a proteolytic enzyme or protease. Scuderi et. al., J. Immunology, 143, 168-173 (1989), suggested that the release of TNF-.alpha. from human leukocyte cells is dependent on one or more serine proteases, e.g., a leukocyte elastase or trypsin. A serine protease inhibitor, p-toluenesulfonyl-L-arginine methyl ester, was found to suppress human leukocyte TNF release in a concentration-dependent manner. Scuderi et. suggested that the arginine methyl ester competes for the arginine-binding site in the enzyme's reactive center and thereby blocks hydrolysis. The lysine and phenylalanine analogs of the inhibitor reportedly failed to mimic the arginine methyl ester.
We have discovered that the protease which causes the cleavage of the TNF-.alpha. molecule into the 17 kD protein is, in fact, a metalloprotease which is believed to reside in the plasma membrane of cells producing TNF-.alpha.. The physicochemical characteristics of the enzyme have not been published.
Most, but not all, proteases recognize a specific amino acid sequence. Some proteases primarily recognize residues located N-terminal of the cleaved bond, some recognize residues located C-terminal of the cleaved bond, and some proteases recognize residues on both sides of the cleaved bond. Metalloprotease enzymes utilize a bound metal ion, generally Zn.sup.2+, to catalyze the hydrolysis of the peptide bond. Metalloproteases are implicated in joint destruction (the matrix metalloproteases), blood pressure regulation (angiotensin converting enzyme), and regulation of peptide-hormone levels (neutral endopeptidase-24.11).
Numerous inhibitors have been developed against the previously described metalloproteases. A general family of inhibitors against matrix-metalloproteases, and in particular collagenase, is reported in WO 92/09563. This document shows compounds having the general structure of a reverse hydroxamate--or a hydroxyurea--linked via an amide to an amino acid derivative, such as tryptophan or 2-naphthyl alanine. Inhibitors of collagenase are also reported in WO 88/06890; these compounds contain sulfhydryl moieties as well as phenylalanine and tryptophan analogs. Collagenase inhibitors are reported in WO 92/09556 and U.S. Pat. No. 5,114,953 and possess hydroxamate moities and fused or conjugated bicycloaryl substituents. The myriad potential gelatinase inhibitors covered by the generic formula in EPA 489,577 are amino acid derivatives optionally possessing a hydroxamate group. Hydroxamate derivatives useful as angiotensin converting enzyme (ACE) inhibitors are reported in EPO 498,665.
Inhibition of the TNF-.alpha. converting enzyme (hereinafter referred to as "TACE"), a novel metalloprotease, inhibits release of TNF-.alpha. into the serum and other extracellular spaces. TACE inhibitors would therefore have clinical utility in treating conditions characterized by over-production or unregulated production of TNF-.alpha.. A particularly useful TACE inhibitor for certain pathological conditions would selectively inhibit TACE while not affecting TNF-.beta. (also known as lymphotoxin) serum levels. The over-production or unregulated production of TNF-.alpha. has been implicated in certain conditions and diseases, for example:
I. Systemic Inflammatory Response Syndrome, which includes:
Sepsis syndrome PA0 Trauma/hemorrhage PA0 Burns PA0 Ionizing radiation exposure PA0 Acute pancreatitis PA0 Adult respiratory distress syndrome. PA0 Post pump syndrome PA0 Ischemia-reperfusion injury PA0 Cardiac stun syndrome PA0 Myocardial infarction PA0 Congestive heart failure PA0 HIV infection/HIV neuropathy PA0 Meningitis PA0 Hepatitis PA0 Septic arthritis PA0 Peritonitis PA0 Pneumonia PA0 Epiglottitis PA0 E. coli 0157:H7 PA0 Hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura PA0 Malaria PA0 Dengue hemorrhagic fever PA0 Leishmaniasis PA0 Leprosy PA0 Toxic shock syndrome PA0 Streptococcal myositis PA0 Gas gangrene PA0 Mycobacterium tuberculosis PA0 Mycobacterium avium intracellulare PA0 Pneumocystis carinii pneumonia PA0 Pelvic inflammatory, disease PA0 Orchitis/epidydimitis PA0 Legionella PA0 Lyme disease PA0 Influenza A PA0 Epstein-Barr Virus PA0 Vital-associated hemaphagocytic syndrome PA0 Vital encephalitis/aseptic meningitis PA0 Premature labor PA0 Miscarriage PA0 Infertility PA0 Rheumatoid arthritis/seronegative arthropathies PA0 Osteoarthritis PA0 Inflammatory bowel disease PA0 Systemic lupus erythematosis PA0 Iridocyclitis/uveitis/optic neuritis PA0 Idiopathic pulmonary fibrosis PA0 Systemic vasculitis/Wegener's granulomatosis PA0 Sarcoidosis PA0 Orchitis/vasectomy reversal procedures PA0 Asthma PA0 Allergic rhinitis PA0 Eczema PA0 Allergic contact dermatitis PA0 Allergic conjunctivitis PA0 Hypersensitivity pneumonitis PA0 ALL PA0 AML PA0 CML PA0 CLL PA0 Hodgkin's disease, non-Hodgkin's lymphoma PA0 MM PA0 Kaposi's sarcoma PA0 Colorectal carcinoma PA0 Nasopharyngeal carcinoma PA0 Malignant histiocytosis PA0 Paraneoplastic syndrome/hypercalcemia of malignancy PA0 Organ transplant rejection PA0 Graft-versus-host disease PA0 Cystic fibrosis PA0 Familial hematophagocytic lymphohistiocytosis PA0 Sickle cell anemia PA0 Psoriasis PA0 Alopecia PA0 Multiple sclerosis PA0 Migraine headache PA0 Nephrotic syndrome PA0 Hemodialysis PA0 Uremia PA0 OKT3 therapy PA0 Anti-CD3 therapy PA0 Cytokine therapy PA0 Chemotherapy PA0 Radiation therapy PA0 Chronic salicylate intoxication PA0 Wilson's disease PA0 Hemachromatosis PA0 Alpha-1-antitrypsin deficiency PA0 Diabetes PA0 Hashimoto's thyroiditis PA0 Osteoporosis PA0 Hypothalamic-pituitary-adrenal axis evaluation PA0 Primary biliary cirrhosis PA0 m is 0, 1 or 2; PA0 R.sup.1, R.sup.2 and R.sup.3 each independent of the other is hydrogen, alkylene(cycloalkyl), OR.sup.4, SR.sup.4, N(R.sup.4)(R.sup.5), halogen, substituted or unsubstituted C.sub.1 to C.sub.8 alkyl, C.sub.1 to C.sub.8 alkylenearyl, aryl, a protected or unprotected side chain of a naturally occurring .alpha.-amino acid; or the group --R.sup.6 R.sup.7, wherein R.sup.6 is substituted or unsubstituted C.sub.1 to C.sub.8 alkyl and R.sup.7 is OR.sup.4, SR.sup.4, N(R.sup.4)(R.sup.5) or halogen, wherein R.sup.4 and R.sup.5 are, each independent of the other, hydrogen or substituted or unsubstituted C.sub.1 to C.sub.8 alkyl; PA0 n is 0, 1 or 2; PA0 provided that when n is 1, A is a protected or an unprotected .alpha.-amino acid radical; PA0 when n is 2, A is the same or different protected or unprotected .alpha.-amino acid radical; and PA0 B is unsubstituted or substituted C.sub.2 to C.sub.8 alkylene; PA0 and the pharmaceutically acceptable salts thereof. PA0 m is 0, 1 or 2; PA0 R.sup.1, R.sup.2 and R.sup.3 each independent of the other is hydrogen, alkylene(cycloalkyl), OR.sup.4, SR.sup.4, N(R.sup.4)(R.sup.5), halogen, substituted or unsubstituted C.sub.1 to C.sub.8 alkyl, C.sub.1 to C.sub.8 alkylenearyl, aryl, a protected or unprotected side chain of a naturally occurring .alpha.-amino acid; or the group --R.sup.6 R.sup.7, wherein R.sup.6 is C.sub.1 to C.sub.8 alkyl and R.sup.7 is OR.sup.4, SR.sup.4, N(R.sup.4)(R.sup.5) or halogen, wherein R.sup.4 and R.sup.5 are each, independent of the other, hydrogen or substituted or unsubstituted C.sub.1 to C.sub.8 alkyl; PA0 n is 0, 1 or 2; PA0 Y is hydrogen, unsubstituted or substituted C.sub.1 to C.sub.8 alkyl, alkylene(cycloalkyl), the group --R.sup.8 --COOR.sup.9 or the group --R.sup.10 N(R.sup.11)(R.sup.12); wherein R.sup.8 is C.sub.1 to C.sub.8 alkylene; R.sup.9 is hydrogen or C.sub.1 to C.sub.8 alkyl; R.sup.10 is unsubstituted or substituted C.sub.1 to C.sub.8 alkylene; and R.sup.11 and R.sup.12 are each, independent of the other, hydrogen or C.sub.1 to C.sub.8 alkyl; PA0 provided that when n is 1, A is a protected or an unprotected .alpha.-amino acid radical; and when n is 2, A is the same or different protected or unprotected .alpha.-amino acid radical; and the pharmaceutically acceptable salts thereof; PA0 wherein the compound is capable of reducing serum TNF-.alpha. levels by at least 80% when administered at 25 mg/kg in a murine model of LPS-induced sepsis syndrome; and a pharmaceutically acceptable carrier.
gram positive sepsis PA1 gram negative sepsis PA1 culture negative sepsis PA1 fungal sepsis PA1 neutropenic fever PA1 urosepsis PA1 meningococcemia
II. Reperfusion Injury, which includes:
III. Cardiovascular Disease, which includes:
IV. Infectious Disease, which includes:
V. Obstetrics/Gynecology, including:
VI. Inflammatory Disease/Autoimmunity, which includes:
VII. Allergic/Atopic Diseases, which includes:
VIII. Malignancy, which includes:
IX. Transplants, including:
X. Cachexia
XI. Congenital, which includes:
XII. Dermatologic, which includes:
XIII. Neurologic, which includes:
XIV. Renal, which includes:
XV. Toxicity, which includes:
XVI. Metabolic/Idiopathic, which includes:
Inhibitors of TACE would prevent the cleavage of cell-bound TNF-.alpha. thereby reducing the level of TNF-.alpha. in serum and tissues. Such inhibitors would be of significant clinical utility and could be potential therapeutics for treating the above TNF-.alpha.-related disorders.